Characterization of Potential Probiotic Lactic Acid Bacteria- Pediococcus

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Journal of Food: Microbiology, Safety f

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ISSN: 2476-2059i 2017, 2:1

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e o J & Hygiene DOI: 10.4172/2476-2059.1000119

Research Article Open Access Characterization of Potential Probiotic Lactic Acid Bacteria- Pediococcus acidilactici Ch-2 Isolated from Chuli- A Traditional Apricot Product of Himalayan Region for the Production of Novel Bioactive Compounds with Special Therapeutic Properties Anupama Gupta* and Nivedita Sharma *Department of Basic Science, Dr. YS Parmar University of Horticulture and Forestry, Nauni, Solan, 173 230, Himachal Pradesh, India *Corresponding author: Anupama Gupta, Department of Basic Science, Dr. YS Parmar University of Horticulture and Forestry, Nauni, Solan, 173 230, Himachal Pradesh, India, Tel: 9821774553; E-mail: [email protected] Received date: March 06, 2017; Accepted date: March 23, 2017; Published date: March 27, 2017 Copyright: © 2017 Gupta A. This is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Objectives: In the present study, Pediococcus acidilactici Ch-2 isolated from Chuli has been evaluated for its probiotic potential and its ability to produce bioactive compounds.

Methodology: Isolation was done on MRS agar followed by the safety assessment of isolates. Isolates were screened on the basis of their antagonistic potential against pathogenic bacteria. Probiotic attributes of screened isolate Ch-2 i.e. acid and bile tolerance, survival in simulated gastrointestinal conditions, bile salt tolerance, auto- and co-aggregation, antimicrobial potential and metabolic profiling have been evaluated.

Results: It was observed that P. acidilactici Ch-2 was resistance to low pH and bile salts (0.3%) and simulated gastric and intestinal conditions, was able to produce bacteriocin and lactic acid against a number of serious food borne and spoilage causing microorganisms. The susceptibility to selected eleven antibiotics, inability to produce gelatinase and DNase and non-hemolytic nature revealed its safe status for further use in food and nutraceutical industry. In the present study, Squalene – a rare and therapeutic anti-cancerous compound has been reported for the very first time from a probiotic bacteria isolated from fermented food product and recommended for its further investigation as a potential source of Squalene.

Conclusion: In conclusion, most of the results in the present work revealed P. acidilactici Ch-2 as a potential probiotic candidate with many functional properties and novel compounds.

Keywords: Chuli; Pediococcus; Probiotics; Himachal Pradesh; acid bacteria as probiotic supplementation has been studied in number Squalene; Lactic acid bacteria of human diseases viz. irritable bowel syndrome, inflammatory bowel diseases, obesity and other allergic diseases. The bioactive compounds Introduction secreted by these bacteria with immunomodulation and therapeutic potential are being utilized more now- a -days due to their Probiotics are defined as live microorganisms that contribute to the effectiveness and safe status [4]. health and well-being of the hosts by maintaining the intestinal microbial balance [1]. Among these, lactic acid bacteria (LAB) are the Use of probiotics with broad antagonistic potential and natural potential probiotic bacteria which are already used in many fermented antimicrobial substances is considered as an appropriate alternative to food products and occur naturally in several raw food materials viz. antibiotic treatment and a better pharmaceutical approach [5]. milk and milk products, meat and flour [2]. LAB is used as natural or Pediococcus acidilactici, a gram positive coccus, cells arranged in selected starters in food fermentations in which they perform tetrad, is a general inhabitant of fermented fruit and vegetable products acidification due to the production of lactic and acetic acids. The and efficiently colonize the digestive tract of the host [6]. Even though protection of food from spoilage and pathogenic microorganisms by the importance of probiotics has been principally coupled with the LAB is related to their antagonistic activity [3]. improvement of gastrointestinal health, recent reports have revealed that potential probiotics also have contributed in reducing other The interest in the microorganisms occurring in foods is primarily metabolic diseases leading to antihypertensive effects [7]. In the due to the biotechnological potential of new bacterial species and present study metabolic fingerprint of the isolate has been elucidated strains. Recently, use of safe and natural supplements instead of to find out the possibility of novel compounds with therapeutic chemotherapeutics has been incorporated because of the advantageous potential and Squalene has been reported for the very first time from and useful effects to the environment. The excessive use of antibiotics probiotic Pediococcus acidilactici (probiotic lactic acid bacteria) and rapid appearance of multiple-antibiotic resistant pathogenic isolated from Chuli with antioxidant properties. bacteria has refocused clinical attention on the field of probiotics and their safe and effective use in the pharmaceuticals. The efficacy of lactic The most important criteria to be considered for the survival and potential benefits on the host are ability to overcome gastric pH and

J Food Microbiol Saf Hyg, an open access journal Volume 2 • Issue 1 • 1000119 ISSN:2476-2059 Citation: Gupta A, Sharma N (2017) Characterization of Potential Probiotic Lactic Acid Bacteria- Pediococcus acidilactici Ch-2 Isolated from Chuli- A Traditional Apricot Product of Himalayan Region for the Production of Novel Bioactive Compounds with Special Therapeutic Properties. J Food Microbiol Saf Hyg 2: 119. doi:10.4172/2476-2059.1000119

Page 2 of 11 the toxic effects of bile salts, the advent in a viable physiological state at probiotics strains. So, antibiotic susceptibility, hemolytic activity, the site of action, should be capable of adhering effectively the Gelatinase and DNase enzyme production were evaluated to ensure intestinal mucosa and coaggregation ability to reduce the ill effects of the safety of the isolate to be used as potential probiotic candidate in pathogens [8]. Most commonly for the isolation of the probiotic food and pharmaceutical industry. bacteria dairy food products have been explored although recently new ideas of isolating new and potential probiotic bacteria from other Tolerance to low acid conditions and bile salts fermented fruit as well as vegetable food products have been adopted worldwide. Chuli is a traditional fermented Apricot product of Acid tolerance of Ch-2 was studied by the method of Liong and Himachal Pradesh with high nutritional profile and a potential source Shah [17] with modifications while tolerance to bile salts was of probiotic lactic acid bacteria (LAB). Therefore, in the present study, determined by Giililand and Walker [18]. Acid tolerance was Pediococcus acidilactici Ch-2 isolated from Chuli has been evaluated determined by comparing the final plate count after 3h with the initial for its probiotic potential and its ability to produce bioactive plate count at 0h. Viability of cells in MRS broth supplemented with compounds with therapeutic potential. 0.3, 1 and 2% bile salts (HiMedia, India) upto 8h was observed by plating 100 µl of culture onto MRS agar plates and incubated at 35°C for 24h. Growth of bacteria was expressed in colony forming units per Materials and Methods milliliter (log CFU/ml) and the percent survival of strain was then calculated. Isolation and identification of probiotic lactic acid bacteria Lactic acid bacteria were isolated from Chuli- a traditional Survival in simulated gastrointestinal conditions fermented Apricot product of Himachal Pradesh for the very first time Survival in simulated gastrointestinal conditions was assessed by for their potentiality as probiotic (Figure 1) using De Man Rogosa and following the method of Charteris et al. [19] with minor modifications. Sharpe (MRS) medium (HiMedia, India) [9]. Chuli (5 g) was mixed An aliquot of 0.1 ml for gastric and intestinal transit assay was with 45 ml of MRS broth and enriched in it for 24 h. Sample was removed after 0, 60 and 240 min. The tolerance was assayed by serially diluted and plated on MRS agar plates at 35°C for 24-48 h determining the viable count in simulated gastric juice after the anaerobically using anaerobic gas packs (HiMedia, India). incubation for different time intervals up to 4h. All the experiments were carried out in triplicates.

BSH activity To evaluate bile salt hydrolase activity, isolate was cultivated in MRS agar containing 0.5 % (w/v) of the sodium salt of taurodeoxycholic acid (TDCA) (HiMedia, India). MRS agar plates were incubated at 35°C for 72 h. The plates were examined for white precipitates which were the sign of bile salt hydrolysis [20].

Autoaggregation Autoaggregation assay was performed according to Del Re et al. [21] with minor modifications. Autoaggregation % was measured as 1- (At/A0) × 100, where At represents the absorbance at time t=1, 2, 3, 4, 5 h and A0 the absorbance at t = 0 h (i.e. 0.5) Figure 1: Chuli- A fermented Apricot product of Himachal Pradesh. Coaggregation Coaggregation ability of the isolate was determined by following the All the isolates obtained after anaerobic incubation were cultivated method described by Del Re et al. [21] with minor modification. in MRS broth and were maintained in 30% glycerol at -20°C. In total 6 Absorbance at λ=600 nm was observed for mixture and each of isolates were obtained and were further tested for Gram reaction [10] individual strain. Co-aggregation % was calculated according to catalase test [11], cell morphology and antimicrobial activity [12]. Handley’s equation [22]. Gram positive, catalase negative isolates were selected and identified to the genus level by their morphological, physiological and biochemical Antimicrobial activity characteristics. Out of six isolates, Ch-2 was selected for further study on the basis of its broadest antagonistic spectrum against spoilage and Antagonistic potential of isolate Ch-2 was evaluated against serious food borne pathogens by using Bit/disc method [12] and was identified food borne as well as spoilage causing microorganisms. Overall to species level by 16S rRNA sequence analysis. antimicrobial activity of non-neutralized cell free supernatant was checked against Listeria monocytogenes MTCC 839, Clostridium Safety assessment perfringens MTCC 1739, Bacillus cereus CRI, Staphylococcus aureus IGMC, Escherichia coli IGMC, Enterococcus faecalis MTCC 2729, Some of the important criteria for the assessment of safe status of Leuconostoc mesenteroides MTCC 107, Pectobacterium carotovorum probiotic bacteria are antibiotic resistance [13], haemolytic activity MTCC 1428 and Pseudomonas syringae IGMC using well diffusion [14] and some enzymes such as Gelatinase [15] and DNase production method [23]. The wells were poured with 150 μL of 24 h old culture [16] and are considered as good indicators in order to select potential

Page 3 of 11 supernatant and the plates were incubated at 35°C for 24 h. The Metabolic fingerprinting antibacterial activity was determined and zones of inhibition were measured in millimeter (mm). Metabolites were extracted by following the method given by Coucheney et al. [26] with minor modifications using GC-MS. Microbial suspensions (20 ml) of isolate (grown in MRS broth for 24 h HPLC- determination of lactic acid at 35°C) were disrupted using pulsed, high frequency sound waves LAB with industrial potential are mostly homofermentative (>20kHz) for five cycles (30 sec. run; one min break) to extract (Pediococcus) which produce only lactic acid as major end product of intracellular metabolites. Suspensions obtained after sonication were their metabolism. Lactic acid, mainly responsible for the antagonistic centrifuged for 10 min at 10,000 rpm in order to separate the extra as potential of isolate Ch-2, was detected by using HPLC (Novapak C-18) well as intracellular solution from the cells. The supernatant was column. Mobile phase used was Methanol: Water (double distilled) removed and the metabolites were extracted with a (95:5) (Sigma Aldrich). Standard organic acid solution i.e. 5% standard methanol:water:chloroform mixture (2:0.8:1):2.5 ml of cold solution of lactic acid (Sigma Aldrich) was prepared in double distilled chloroform and 5 ml of cold methanol (-20°C) were added to and the water. Twenty four hour old culture grown in MRS broth was phases were allowed to separate. Metabolic fingerprints were measured centrifuged to get the culture supernatant. Sample was filtered using a in the aqueous phase after freeze drying while in the organic phase; the 0.22 µm Millipore filter membrane before using for analysis. HPLC dried chemical extracts obtained after complete evaporation of the analysis was firstly performed with standard organic solution followed solvent was used for metabolic profiling. by the samples. The monitoring was done at 210nm. Results Production of bacteriocin An overnight culture of Ch-2 was inoculated (OD600 = 1, 10%) into Isolation and identification 100ml of MRS broth and cultivated at 37°C for 24 h. Samples were Samples of Chuli taken from various parts of Himachal Pradesh taken every 2 hours for 48 h and bacteriocin activity was measured. To were used as isolation source. Lactic acid bacteria were isolated on quantify the bacteriocin activity, the agar well diffusion method was MRS agar at 37°C under anaerobic conditions. Six lactic acid bacterial used. Neutralized cell free culture supernatant (NCFS) was serially isolates were obtained from Chuli and 4 out of 6 were confirmed as diluted twofold in sterile distilled water and 150μl of each dilution was rods while two were confirmed as coccus by microscopic examination. added into the wells. Activity units of bacteriocin production were All the isolates were Gram positive, catalase negative, unable to utilize estimated as AU/ml, where AU/ml is defined as the reciprocal of the citrate, unable to hydrolyze casein and no urease and indole highest dilution that resulted in inhibition of the indicator strains. L. production was observed by any of the isolates. On the basis of monocytogenes was used as a bacteriocin sensitive indicator strain to morphological and biochemical characteristics, isolates were determine bacteriocin activity levels [24]. tentatively identified at genus level as Lactobacillus and Pediococcus (tetrad). Out of six isolates, Ch-2 gave clear zones around the lawn of H2O2 production indicator baterial strains using bit/disc method with broadest antimicrobial spectrum and was selected for further study. The largest Quantitaive estimation of Hydrogen Peroxide (H O ) was done by 2 2 diameter of inhibition (up to 26.6mm) was obtained against L. following the method given in AOAC [25]. Suspension was titrated monocytogenes, C. perfringens, B. cereus and P. syringae (Figure 2). against 0.1 N KMnO4 (Sigma Aldrich) until the suspension becomes colorless. Each ml of 0.1 N KMnO4 is equivalent to 1.070 mg of H2O2.

Figure 2: Antagonistic spectrum of lactic acid bacteria isolated from Chuli by Bit/disc method against bacterial indicators.

After screening, DNA of the isolate was subjected to PCR for The PCR products obtained after amplification were visualized using amplification of small subunit of 16S rRNA genes using universal ethidium bromide (Thermo Fisher Scientific) on 1.5% agarose gel primers (BITS-1(5’AGAGTTTGATCCTGG) and BITS-4-(5’- (Sigma-Aldrich). These after purification had got sequenced by TACCTTGTTACGACTT) having expected product size of 1500bp. Xceleris, India Pvt. Ltd. to identify the isolate. Sequence similarity

Page 4 of 11 search for the isolate Ch-2 (BLAST, NCBI) showed 99% similarity with Safety assessment of P. acidilactici Ch-2 the available sequence of Pediococcus acidilactici and was assigned accession no KJ541886 by NCBI. Phylogenetic tree of Pediococcus P. acidilactici Ch-2 was found to be sensitive to 11 out of 12 acidilactici Ch-2 with respect to other lactic acid bacteria along with antibiotic (with 91.66% sensitivity) used in the study (Table 1), thereby the boot strap values as inferred by the neighbor joining method advocating its inability to grow in the presence of antibiotics and thus (Mega 6) has been presented in Figure 3. its safe status. Isolate showed a negative response in the production of DNase and gelatinase enzymes as pathogenicity factors. No clear zones around colonies were observed on blood agar medium (Figure 4). Haemolytic activity would break down the epithelial layer while the gelatinase activity would damage the mucoid lining. Absence of haemoloytic and gelatinase activity is a selection criterion for probiotic strains, indicating that this bacterium is non-virulent [27].

Tolerance to low pH and bile salts In the present investigation, Ch-2 was subjected to as low pH as 1 (during fasting) and was found to survive it for about 2 h with 74.95% survival. Under acidic conditions of pH 2 and 3, isolate Ch-2 showed resistance upto 3 h with 72.69 and 98.62% survival, respectively (Figure 5). Probiotic bacteria require to survive gastric passage, where the pH can be as low as 1.5 to 2.0 [28], and stay alive for 4 h or more [29] before they move to the intestinal tract. As the isolate in the present investigation was able to survive gastric pH (even as low as 1) upto 3h, therefore it can be considered as a good probiotic and can be Figure 3: Neighbour-joining phylogenetic dendrogram of strain recommended in the food preparations for its effective delivery in the Ch-2 and related lactic acid bacterial species based on 16S rRNA system. gene sequence similarity. Numbers at branch points are levels of Isolate Ch-2 was able to survive the required concentration of 0.3% bootstrap support (%) based on analysis of 1000 re-sampled bile salts with 86.7% survival when evaluated through optical density datasets. method. Cell viability of isolate Ch-2 upto 8 h was reported to be 98.34%, 96.40% and 95.42% survival for 0.3, 1.0 and 2.0% bile salt concentration, respectively (Figure 6).

S. No Antibiotics Concentration (µg) *S/R

1. Ampicillin (AMP) 30 S

2. Augmentin (AMC) 30 S

3. Gentamicin (GEN) 10 S

4. Cephalothin (CEP) 30 S

5. Cloxacillin (COX) 1 S

6. Cefotaxime (CTX) 30 S

7. Cefoxitin (CX) 30 S

8. Lincomycin (L) 2 S

9. Tetracycline (TE) 30 S

10. Amoxyclav (AMC) 30 S

11. Co-trimoxazole (COT) 25 R

12. Cefuroxime (CXM) 30 S

% Sensitivity 91.66

Table 1: Antibiotic sensitivity of P. acidilactici Ch-2. *Sensitive, Resistant.

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Figure 4: DNase, gelatinase and hemolytic activities of P. acidilactici Ch-2.

survival for 1h and 4h, respectively. The data obtained reveal that the isolate was efficient in surviving the harsh conditions of gastrointestinal conditions, thus can be used for further study and recommended for its use as successful probiotic.

BSH activity Survival under simulated gastrointestinal conditions indicates the resistance of the selected strain to bile salts, which could be ascribed to bile salt hydrolase (BSH) activity. BSH catalyses the deconjugation of bile salts and free (deconjugated) bile salts resulted after the deconjugation have lower solubility at low pH and thus, precipitate as a Figure 5: Tolerance of P. acidilactici Ch-2 to low acidity. result of the fermentative metabolism of LAB [30]. But in our investigation, isolate Ch-2 was unable to deconjugate the bile acids (no growth of cells was observed on plates) though it exhibited good survival under high concentration of bile salts.

Autoaggregation and Coaggregation The autoaggregation ability of isolate Ch-2 was determined during a period of 5 h. Autoaggregation percentage of bacterial isolate was measured by comparing the initial absorbance at 600 nm with absorbance of 1st, 2nd, 3rd, 4th and 5th h of incubation. It was found that the cells of isolate Ch-2 efficiently aggregated (97.0% aggregation) with each other over a period of 5 h. The minimum required autoaggregation percentage for good probiotic strains has been recommended more than 40% as the time proceeds more than 5 h Figure 6: Tolerance of P. acidilactici Ch-2 to Bile salt concentrations. [14]. As depicted by the results described above, isolate Ch-2 exhibited strong autoaggregation potential as the strain has autoaggregation Survival in simulated gastrointestinal conditions more than 40% over a period of 5 h Ch-2 has a coaggregation percent of 20.83, 16.32% and 6.25 with L. monocytogenes, C. perfringens and Isolate Ch-2 when exposed to gastric juice (pH 2), exhibited 74.32% B. cereus (Figure 7). As the isolate showed maximum coaggregation % survival upto 1h while percent survival at pH 3 was found to be 77.22 with L. monocytogenes, a serious food borne pathogen, it depicts its and 57.18% for 1 and 4 h, respectively (as shown in Table 2). Resistance ability to inhibit the serious pathogen to the maximum extent. to intestinal transit was studied at pH 8.0 and found that the isolate Ch-2 successfully survived the intestinal juice with 96.13 and 79.50%

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Survival percentage (%)

Gastrointestinal juices 1h 4h

log CFU/ml % Survival log CFU/ml % Survival

Pepsin (pH 2) 7.7(± 0.20) 62.0 - -

Pepsin (pH3) 8.0(± 2.00) 64.51 5.97(± 0.02) 48.1

Pancreatin (pH8) 10.08 (± 0.04) 81.29 8.3 (± 0.20) 66.90

Table 2: Resistance of isolate Ch-2 to simulated gastrointestinal juices. *Log cfu/ml: Mean (± SD) of results from three separate experiments. **% Survivability = (log cfu 4th, 1st h / log cfu 0th h) × 100.

HPLC- determination of Lactic acid P. acidilactici Ch-2 isolated from Chuli was able to inhibit the tested indicator organisms with varied zones of inhibition. The inhibitory spectrum of strains against pathogenic bacteria is shown in Fig 9. Some species of Staphylococcus aureus, Enetrococcus, Leuconostoc and Listeria are highly pathogenic to human beings. The isolated P. acidilactici Ch-2 showed strong bactericidal activity against these species. The pH and titratable acids in the cell free culture supernatant of P. acidilactici Ch-2 were 3.63 and 0.07%, respectively. Pediococcus are homofermentative bacteria which produce more than 85% lactic acid from glucose being the major product of fermentation. P. acidilactici Ch-2 being a homofermentative culture was evaluated for lactic acid production which was confirmed by HPLC analysis Figure 7: Coaggregation percent of P. acidilactici Ch-2. (Novapak C-18) and has been found to be 7.911 mg/L after 24 h of incubation. Figure 8 shows typical HPLC chromatograms of standard and lactic acid extracted from culture supernatant of P. acidilactici Ch-2 revealing its antimicrobial potential Figure 9. Antimicrobial activity The antagonistic potential of Ch-2 was assessed against most challenging and serious food borne pathogens. The inhibitory spectrum of isolate Ch-2 has been described in Figure 8. Isolate Ch-2 exhibited a broad spectrum of inhibition against test pathogens with zones of inhibition ranging from 17.6-23.6 mm. Efficacy and effectiveness of antimicrobial potential of Pediococcus spp. have been studied widely.

Figure 9: HPLC Chromatogram of a) standard solution of lactic acid b) culture supernatant of P. acidilactici Ch-2.

Production of bacteriocin The antagonistic pattern of P. acidilactici Ch-2 was evaluated against test pathogen viz. L. monocytogenes and has been found to inhibit it efficiently (Figure 10).

Figure 8: Antagonistic spectrum of P. acidilactici Ch-2. H2O2 production

Probiotic isolate P. acidilactici was evaluated for H2O2 production and has been reported to produce 0.61g/L.

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Metabolic fingerprinting Tetradecane (2.11%) and Dodecane (1.73%) possess good antioxidant properties. Two rare compounds with therapeutic potential have been The metabolite composition of P. acidilactici Ch-2 provides a reported to be produced by this isolate for the very first time from any window for elucidating its safety and possibility of finding novel probiotic bacteria i.e. 9Octadecenamide, (Z) (6.06%) and Squalene compounds with nutritive as well as health properties. In the current (0.54%) (Figure 11). study, intra- as well as extracellular metabolites were assessed by GC- MS analysis and the isolate revealed the presence of 41 compounds 9 Octadecenamide, (Z) has a therapeutic potential and is having a (four were obtained in aqueous fraction while 38 were observed in strong activity against sleep and mood related disorders. Squalene, in solvent fraction and 1compound was common to both) of which 14 addition to have antimicrobial activity, has also been reported to have compounds were found to have biological activities. These compounds anticancer, anti-oxidant, chemo-preventive, anti-tumor and sunscreen were confirmed based on their peak area, retention time and molecular properties [31]. Squlene, a triterpine compound, was originally found formula and are represented in Table 3a and b. in shark liver oil. It can also be obtained from plant sources; however the production at the desired purity level for food and pharmaceutical applications cannot be achieved. The use of microbial production of squalene may be a suitable alternative to address the issue. Currently, Saccharomyces cerevisiae has been used successfully for its production while other food grade probiotic lactic acid bacteria or other bacilli have not been reported so far to produce squalene [32,33]. This study becomes of paramount importance by revealing the production of Squalene from probiotic bacteria 1st time and can open a gateway for assessing the different probiotics for unique bioactive compounds of special pharmacy properties. Thus potential of this isolate for Squalene production can be exploited by optimizing the yield and can further be used for commercial production of Squalene along with its good probiotic capabilities.

S. Compou Ret Molecul Peak Are Peak height Biological No. nd Name enti ar Area a % activity on Formul Tim a e

1 L-Lactic 6.13 C3H6O 182319 85.1 80403220.7 Antimicrobi acid 3 8239.01 9 9 al

Figure 10: Bacteriocins production by P. acidilactici Ch-2. 2 Pyrrolo[1, 13.3 C7H10N 126120 5.89 9336208.17 Antimicrobi 2a] 9 2O2 542.69 al, pyrazine1 antioxidant ,4dione, The compounds identified using GC-MS possessed many biological hexahydr activities helpful in food industry as well as in pharmaceutical industry o with therapeutic potential such as L-Lactic acid (85.19%), 3 Dibutyl 14.6 C16H22 610851 2.85 8439561.87 - Methyltartronic acid (7.47%), Pyrrolo [1,2a] pyrazine1,4dione, phthalate 0 O4 91.70 hexahydro (5.89%) and Phenol, 2,4bis (1,1dimethylethyl) (8.32%) exhibited good antibacterial activity. n-Hexadecanoic acid (6.61%) 4 *9Octade 17.5 C18H35 129710 6.06 16525865.5 Anti-sleep possesses anti-inflammatory, Geranyl-isovalerate (0.33%), cenamide 2 NO 489.48 3 disorder , (Z) agent Benzaldehyde, 2,4 dimethyl (1.23%) and Hexadecane, 2,6,11,15 tetramethyl (0.71%) were reported to be flavor agents and are being used as food additives. Hexadecane (2.00%), Pyrrolo [1,2a] pyrazine1,4 Table 3a: Metabolic profile of P. acidilactici Ch-2 (aqueous fraction). dione, hexahydro3 (2methylpropyl) (0.72%), Hexadecane (1.70%),

S. No. Compound Name Retent Molecular Peak Area Area % Peak height Biological ion Formula activity Time

1 Trichloromethane 3.09 CHCl3 2151864601.56 13.80 377516301.26 -

2 Butane, 2ethoxy2methyl 3.56 C7H16O 1296781662.84 8.32 101201149.67 -

3 Diethyl carbonate 4.29 C5H10O3 1716108545.09 11.01 202279957.97 -

4 Butane, 2,3dichloro2methyl 4.68 C5H10Cl2 125154855.13 0.80 14275282.52 -

5 Benzene, chloro 5.01 C6H5Cl 893423584.87 5.73 82472224.20 -

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6 Methyltartronic acid 5.96 C4H6O5 1164627846.18 7.47 89720023.58 Antibacterial

7 Cyclopentane, 1,3dichloro, cis 6.63 C5H8Cl2 484275080.34 3.11 49194324.93 -

8 Propanal, 2,3dichloro2methyl 7.30 C4H6Cl2O 400387071.13 2.57 41587372.49 -

9 Undecane 7.98 C11H24 183319732.83 1.18 33643465.46 -

10 Benzoic acid 8.87 C7H6O2 139633781.42 0.90 21482083.85 -

11 Dodecane 9.02 C12H26 269670925.56 1.73 59154152.22 Antioxidant

12 Benzaldehyde, 2,4dimethyl 9.31 C9H10O 192127881.29 1.23 41299139.71 Flavor agent

13 Benzene, 1,3bis(1,1dimethylethyl) 9.62 C14H22 647249953.19 4.15 169956544.53 -

14 Trichloroacetic acid, hexadecyl 10.07 C18H33Cl3O2 68316277.38 0.44 7818007.08 - ester

15 Dodecane, 2,6,11trimethyl 10.23 C15H32 84724504.08 0.54 14572730.71 -

16 Tetradecane 10.90 C14H30 329137555.64 2.11 78166898.57 Antioxidant

17 Phenol, 2,4bis(1,1dimethylethyl) 11.92 C14H22O 1297998762.54 8.32 318041677.69 Antimicrobial

18 Hexadecane, 2,6,11,15tetramethyl 12.10 C20H42 111256301.00 0.71 20177977.66 Natural food additive

19 Dodecanoic acid 12.33 C12H24O2 119319719.97 0.77 19561701.25 -

20 Hexadecane 12.55 C16H34 265278473.56 1.70 60185485.26 Antioxidant

21 Geranyl isovalerate 12.94 C15H26O2 51700961.39 0.33 6917915.01 Flavor agent

22 Tetradecane, 2,6,10trimethyl 13.33 C17H36 77962782.28 0.50 11468334.41 -

23 Tetradecanoic acid 13.85 C14H28O2 282944336.98 1.81 20902215.25 -

24 10Methylnonadecane 14.05 C20H42 295915206.74 1.90 39204553.25 -

25 iPropyl 12methyltridecanoate 14.24 C17H34O2 50533687.90 0.32 13277202.93 -

26 Pyrrolo[1,2a]pyrazine1,4dione, 14.56 C11H18N2O2 111498511.60 0.72 10373887.81 Antioxidant hexahydro3( 2methylpropyl)

27 n-Hexadecanoic acid 15.23 C16H32O2 1030968696.76 6.61 80644345.90 Anti-inflammatory

28 Z5Methyl6heneicosen11one 16.19 C22H42O 166958679.19 1.07 11246215.74 -

29 Eicosanoic acid 16.69 C20H40O2 349862423.03 2.24 19708800.55 -

30 1Hexadecanol, 2methyl 16.98 C17H36O 83780395.92 0.54 9128309.20 -

31 *9Octadecenamide,(Z) 18.17 C18H35NO 395784730.00 2.54 48534273.34 Anti-sleep disorder agent

32 Pyrrolo[1,2a] pyrazine1,4dione, 18.61 C14H16N2O2 125921545.39 0.81 10456092.22 - hexahydro3(phenylmethyl)

33 Tetratriacontane 20.16 C34H70 37940689.16 0.24 4004069.70 -

34 Phthalic acid, di(2propylpentyl) 20.31 C24H38O4 53198945.89 0.34 8477434.89 - ester

35 Tetracosane 20.90 C24H50 40257312.22 0.26 4304905.66 -

36 Hexadecanoic acid, hexadecylester 24.00 C32H64O2 64912603.62 0.42 3021684.96 -

37 Octadecane, 3ethyl5(2ethylbutyl) 25.16 C26H54 67973082.92 0.44 4149687.97 -

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38 Squalene 25.89 C30H50 53908277.01 0.35 7539688.23 Anti-cancerous

Table 3b: Metabolic profile of P. acidilactici Ch-2 (organic solvent fraction). *Common compounds.

Klare et al. [39] studied the antibiotic susceptibility of Pediococcus pentosaceus (20), Pediococcus acidilactici (29), Pediococcus sp. (49) and Lactococcus lactic (8) and found that none of the pediococci and lactococci tested showed acquired resistance to antibiotics used in the study viz. Streptomycin, Vancomycin, Erythromycin, Clindamycin, Oxytetracyclin and Fusidic acid. Similarly, Cota-Gastelum et al. [40] studied haemolysis pattern of LAB isolated from fish and found that out of 37 presumptive Pediococcus pentosaceus and Pediococcus spp., 26 presented β-haemolysis and 11 presented gamma haemolysis. Gamma haemolysis is displayed by bacteria that do not induce haemolysis of the blood cells while isolates with beta haemolysis were not characterized because of their capacity to lyse human and tilapia erythrocytes. The most important factors that may affect the survival of probiotics are the high acidity in the gastric region and the high concentration of bile components in the proximal intestine [41]. Therefore, during the selection of potential probiotic isolates to assure their viability and Figure 11: GC-MS chromatogram of Squalene- a novel compound functionality being tolerant to acidic conditions is an important secreted by P. acidilactici Ch-2. criterion to be considered. Moreover, most of the probiotic bacteria exhibit unpredictable resistance to acidic conditions and this characteristic is species and strain dependent [42]. Probiotic bacteria Discussion should pass through the highly acidic stomach in order to reach the intestine and create proper conditions for residence. Fermented food products have a long history and they form a significant part of the diet of many indigenous communities in the Generally, the tolerance to bile salts has been considered a condition developing world. Recently, there has been an increase of interest for survival, colonization and metabolic activity of probiotic bacteria in regarding the commercial utilization of probiotic LAB strains isolated the host’s intestine. As the bile salts can influence the intestinal from traditional and naturally fermented food products which possess microflora by acting as antimicrobial molecules, consequently, it is of medicinal and health-promoting effects. In addition to fermented food immense importance to evaluate their ability to tolerate bile salts [41]. products, microbial flora of traditional fresh food sources such as fruits The ability to survive gastrointestinal transit in high viable cell counts and vegetable samples with high nutritional as well as therapeutic is an important probiotic criterion. Selection of potential probiotic characteristics have also been explored to study their potential in food strains which could survive in the GI tract was performed on the basis and pharmaceutical sector. Sadrani et al. [34] also used fruits in of the measurements of strain survival in simulated gastric (pH 2,3) addition to nine samples of fermented foods (cheese, curd and and intestinal juices (pH 8). shrikhand) for the isolation of LAB using De Man Rogosa and Sharpe The autoaggregation ability is one of the key factors that determine (MRS) broth. Probiotics lactic acid bacteria have been shown to the ability of the probiotic strain to adhere to the oral cavity, suppress pathogenic growth through the release of a variety of gastrointestinal tract and urogenital tract and coaggregation ability antimicrobial factors viz. bacteriocins, H2O2, ammonia, diacetyl and helps to from a barrier that prevents colonization by pathogens. organic acids such as lactic and acetic acids, which reduce the pH of Probiotics are able to do coaggregation with pathogens will effectively the lumen, resulting in the inhibition of the growth of a variety of inhibit and kill pathogenic bacteria as antimicrobial compounds food-borne spoilage and pathogenic organism [35]. By producing produced can move directly on pathogens [43]. The antimicrobial these antimicrobial compounds, probiotic microorganisms gain an action of probiotic bacteria is reportedly due to their potential to edge over other microorganisms to survive in the adverse conditions of produce lactic acid, H2O2 and antimicrobial peptides (bacteriocins) gastrointestinal tract [36]. Similarly, Gautam et al. [37] studied the [44]. Mandal et al. [45] studied the antagonistic potential of P. antagonistic potential of lactic acid bacteria isolated from Dulliachar-a acidilactici LAB5 against some food spoilage and human pathogenic salted pickle and found to exhibit broad spectrum activity against bacteria and proved the antagonistic efficacy of antibacterial substance spoilage causing/food borne pathogens viz. L. monocytogenes, C. secreted by the strain i.e. bacteriocin against pathogenic bacteria viz. perfringens, S. aureus, L. mesenteroides, L. plantarum and B. cereus. Listeria, Staphylococcus, Streptococcus, Enterococcus and A vital issue during the selection and evaluation of new probiotics is Leuconostoc. P. pentosaceus 2A2 and 1A6 isolated from Indonesian their safety analysis. Thus, characterization of the safety criteria of the local beef exhibited antibacterial activity against E. coli ATCC25922, putative probiotic strains is obligatory in order to avoid their side EPEC, S. typhimurium ATCC14028 and S. aureus ATCC25923 effects [38]. Among them, some such as antibiotic susceptibility, (inhibition zones 5.5-14mm) [46]. haemolytic, gelatinase and DNase production testing have been ranked The antimicrobial peptides produced by Pediococcus spp. are the must to ensure safety and non-virulent nature of probiotic bacteria. categorized in the 2nd class of bacteriocins from LAB and are known as

Page 10 of 11 antilisterial bacteriocins. In general class IIa bacetriocins have a rather References narrow spectrum of activity [47,48]. All class II bacteriocins are described as being active against Listeria. Hydrogen peroxide is 1. Asahara T, Shimizu K, Nomoto K, Hamabata T, Ozawa A, et al. (2004) Probiotic bifidobacteria protect mice from lethal infection with Shiga normally produced by vaginal lactobacilli isolates, but may also be toxin-producing Escherichia coli O157:H7. Infect Immun 72: 2240-2247. associated with intestinal lactobacilli or those living in the 2. Rodríguez E, González B, Gaya P, Núñez M, Medina M (2000) Diversity environment [49,50]. This trait is rarely reported in probiotic LAB of bacteriocins produced by lactic acid bacteria isolated from raw milk. isolated from fermented food products. In this study, Pediococci were Int Dairy J 10: 7-15. first time reported to produce H2O2 as an antimicrobial agent against 3. Parvez S, Malik KA, Ah Kang S, Kim HY (2006) Probiotics and their food spoilage pathogens. Thus, this trait may be beneficial in fermented food products are beneficial for health. J Appl Microbiol 100: improving vaginal health and in preventing urinogenital infections. On 1171-1185. the basis of the results, it can be hypothesized that the antagonistic 4. Vitali B, Ndagijimana M, Cruciani F, Carnevali P, Candela M, et al. (2010) activity of the strain relies on acidity, lactic acid, H2O2 and other Impact of a synbiotic food on the gut microbial ecology and metabolic antimicrobial compounds (bacteriocins), thus revealing its potential profiles. BMC Microbiol 10: 4. and safe use as bio preservative agent in food and fermentation 5. Ahmadi S, Soltani M, Shamsaie M, Islami HR, Peyghan R (2014) industry. Comparative effect of Pediococcus acidilactici as probiotic and vitamin C on survival, growth performance and enzyme activities of While Leg Broad spectrum antagonism by probiotic microorganism through Shrimp (Litopenaeus vannamei). J Anim Vet Adv 13: 877-885. various antimicrobial substances especially bacteriocin production is 6. Klaenhammer TR (1993) Genetics of bacteriocins produced by lactic acid currently well accepted phenomenon [51] as compared to older bacteria. FEMS Microbiol Rev 12: 39-85. concept where bacteriocin inhibition was linked against closely related 7. Lye HS, Kuan CY, Ewe JA, Fung WY, Liong MT (2009) The improvement species only [52-54]. There are many evidences reporting the secretory of hypertension by probiotics: effects on cholesterol, diabetes, renin, and antibacterial components produced by LAB having broad range phytoestrogens. Int J Mol Sci 10: 3755-3775. antagonism against Gram-positive and Gram-negative organisms [55] 8. Nur YZ, Aslim B (2010) Assessment of potential probiotic and starter which are independent of lactic acid and hydrogen peroxide. properties of Pediococcus spp. isolated from Turkish-type fermented sausage (Sucuk). J Microbiol Biotechnol 20: 161-168. Probiotics play a major role in health and wellbeing beyond basic 9. De Man J, Rogosa M, Sharpe M (1960) A medium for the cultivation of nutrition. Applications of probiotic bacteria and their metabolites in lactobacilli. J Appl Bacteriol 3: 130-135. pharmaceutical preparation, medicines, food products and dietary 10. Gram HC (1884) Über die isolierte Färbung der Schizomyceten in supplements are increasing due to their potential health benefits, such Schnitt- und Trockenpräparaten. Fortschritte der Medizin 2: 185–189. as antimicrobial activity, antimutagenic, anticarcinogenic activities, 11. Aneja KR (2003) Experiments in Microbiology, Plant pathology and regulation of immune function reducing lactose intolerance, Biotechnology. Biochemical activities of microorganisms, 4th edn. New constipation, improving gastrointestinal health and allergenic diseases age International Publishers, New Delhi, pp. 245-275. such as food allergy, etc. [56-58]. These properties of probiotic lactic 12. Barefoot SF, Klaenhammer TR (1983) Detection and activity of lactacin acid bacteria have been explained by the production of different B, a bacteriocin produced by Lactobacillus acidophilus. Appl Environ Microbiol 45: 1808-1815. primary and secondary metabolites that exert their biological effects Thirabunyanon M, Boonprasom P, Niamsup P (2009) Probiotic potential either directly or by modulating the immune system. The reliability of 13. of lactic acid bacteria isolated from fermented dairy milks on the methodology in assessing the impact of metabolic compounds on antiproliferation of colon cancer cells. Biotechnol Lett 31: 571–576. colonic metabolic signature has been recently demonstrated [4,59,60]. 14. Harrigan WF (1998) Laboratory methods in food microbiology. Academic Press: New York. Conclusions 15. Harrigan WF, McCance ME (1990) Laboratory Methods in Food and Dairy Microbiology. Academic Press, London. Pediococcus acidilactici Ch-2 isolated from Chuli has been 16. Gupta H, Malik RK (2007) Incidence of virulence in bacteriocin evaluated for its probiotic potential and was found resistance to low pH producing enterococcal isolates. Lait 87: 587 601. and bile salts (0.3%) and simulated gastric and intestinal conditions, 17. Liong MT, Shah NP (2005) Acid and bile tolerance and cholesterol was able to produce bacteriocin and lactic acid against a number of removal ability of lactobacilli strains. J Dairy Sci 88: 55-66. serious food borne and spoilage causing microorganisms. The 18. Gilliland SE, Walker DK (1990) Factors to consider when selecting a susceptibility to selected eleven antibiotics, inability to produce culture of L. acidophilus as a dietary adjunct to produce a gelatinase and DNase and non-hemolytic nature revealed its safe status hypercholesterolemic effect in humans. J Dairy Sci 73: 905-909. for further use in food and nutraceutical industry. In the present study, 19. Charteris WP, Kelly PM, Morelli L, Collins JK (1998) Development and Squalene – a rare and therapeutic anti-cancerous compound has been application of an in vitro methodology to determine the transit tolerance reported for the very first time from a probiotic bacteria isolated from of potentially probiotic Lactobacillus and Bifidobacterium species in the fermented food product and recommended for its further investigation upper human gastrointestinal tract. J Appl Microbiol 84: 759-768. as a potential source of Squalene. In conclusion, most of the results in 20. Dashkevicz MP, Feighner SD (1989) Development of a differential medium for bile salt hydrolase-active Lactobacillus spp. Appl Environ the present work revealed P. acidilactici Ch-2 as a potential probiotic Microbiol 55: 11-16. candidate with many functional properties and novel compounds. 21. Del Re B, Sgorbati B, Miglioli M, Palenzona D (2000) Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium Conflict of Interest longum. Lett Appl Microbiol 31: 438-442. 22. Handley PS, Harty DW, Wyatt JE, Brown CR, Doran JP, et al. (1987) A Conflict of interest declared none. comparison of the adhesion, coaggregation and cell-surface hydrophobicity properties of fibrillar and fimbriate strains of Streptococcus salivarius. J Gen Microbiol 133: 3207–3217.

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